Antifungal susceptibility patterns of colonized Candida species isolates from immunocompromised pediatric patients in five university hospitals.

Background and Objectives: Colonization of Candida species is common in pediatric patients admitted to hematology-oncology wards. The aim of this study was to identify colonized Candida species and their susceptibility patterns in hematologic pediatric patients. Materials and Methods: Samples were collected from mouth, nose, urine and stool of the patients admitted to five university hospitals and cultured on sabouraud dextrose agar. The isolates were identified by API 20 C AUX system and their susceptibility patterns were evaluated by CLSI M27-A3 and S4. Results: From 650 patients, 320 (49.2%) were colonized with 387 Candida species. Candida albicans was the most prevalent isolated species, followed by Candida glabrata, Candida tropicalis, Candida famata, Candida kefyr and Candida kuresi. The epidemiological cut off value (ECV) for all Candida species to amphotericin B was ≤0.25 μg except C. krusei (4 μg). The resistance rate to fluconazole in this study in C. albicans was 4.9% with ECV 8 μg/ml, followed by C. tropicalis 8.8% with ECV 0.5 μg/ml. Voriconazole and posaconazole were effective antifungal agents for all Candida isolates. The ECV of C. albicans, Candida parapsilosis, C. tropicalis, C. glabrata and C. krusei for itraconazole were 0.5, 0.25, 0.5, 1 and 2 μg, respectively. The resistant and intermediate rates of Candida species to caspofungin in this study were 2.9%, 5.9%, 18.8%, 47.9%, 0.0% and 16.7% in C. tropicalis, C. glabrata and C. parapsilosis respectively. Conclusion: C. albicans was the most prevalent species in pediatric colonized patients. New azole agents like voriconazole and posaconazole are effective against non-albicans Candida species. Increase in intermediate species is alarming to future emerging resistant species.

Sample collection. The present study was con-ducted from 2014 to 2015 in order to investigate the fungal colonization from immunocompromised children admitted to five university hospitals in Iran (Shiraz, Kerman, Yasouj, Ahvaz and Sannandaj). Totally, 1950 samples were collected from mouth, nose, urine and anus. Samples were cultured on Sabouraud dextrose agar (Merck, Darmstadt, Germany) and transferred to Prof. Alborzi Clinical Microbiology Research Center for further examination. To evaluate the purity of isolates, the samples were cultured on potato dextrose agar (OXOID LTD, Basind stoke, Hampshire, England) twice at 35ºC for 48h. The isolates were identified by carbohydrate assimilation reactions on API 20 C AUX system (bioMerieux, Swiss), according to the manufacturer's instructions.
Antifungal susceptibility testing. The susceptibility patterns of the isolates against amphotericin B, fluconazole, ketoconazole, voriconazole, itraconazole, caspofungin and posaconazole (GmbH-Steinheim-SiGMA-Aldrichmie) were investigated using broth micro dilution assay, according to CLSI M27-A3 and S4 guidelines (9,10). C. parapsilosis ATCC22019 and C. krusei ATCC6258 were considered as standard strains. The final concentrations of amphotericin B, itraconazole, posaconazole and voriconazole were ranged from 0.032 to 16 μg/ml and for fluconazole and caspofungin from 0.125 to 64 μg/ml and 0.016 to 8 μg/ml, respectively. In each series, one negative control without any yeast suspension and one positive control without any drugs were considered. The plates were sealed and incubated for 24 and 48h at 35ºC and visual minimum inhibitory concentration (MIC) end points were determined. The recommended end-point for azole and caspofungin are the lowest drug concentration with a prominent decrease in turbidity (inhibitory concentration that gives 50% growth reduction), while for amphotericin B, MIC was the drug concentration showing a complete inhibition of growth. According to CLSI M27-A3 and S4, there is not any breakpoint for posaconazole and ketoconazole (9, 10).

Statistical analysis.
Statistical analysis was performed using WHO NET (version 5.6). Epidemiological cutoff value (ECV), Wild-type (WT) and non-WT strain, MIC50 and MIC90 value and Geometric Mean (GM) were reported.

diSCUSSiON
For the management of systemic candidiasis in immunocompromised patients, early diagnosis and empirical antifungal therapies are in focus. Leon et al. reported multifocal colonization (OR=3.04, 95% CI, 1.45-6.39) was predictive of proven Candida infection and would benefit from early antifungal therapy (11). As the colonized Candida may transfer to pathogen due to change in patients' immune system, knowl- There is no breakpoint, only MIC was reported.
There is no breakpoint, only MIC was reported.
S: Susceptible, SSD: Susceptible dose dependent, I: Intermediate, R: Resistant, ECV: Epidemiological cut off value; WT: Wild type, NWT: Non-wild type rates of non albicans Candida bloodstream infection in children were reported 29.7%, 41.7% and 57.1% for C. parapsilosis, C. tropicalis and C. glabrata, respectively (19). Distributions of Candida species are different according to region and patient's populations. Therefore, identification of Candida species isolated from pediatric patients is valuable in each region. Amphotericin B is a common antifungal agent recommended for fungal infection therapy but its use has some limitations due to the risk of toxicity. In the present study, most of Candida species isolates were susceptible to amphotericin B except C. albicans and C. krusei with resistance rates of 0.9% and 38.5%, respectively.
Fluconazole is a triazole agent that is the most prescribed antifungal agents for the treatment of Candida infections. Other azoles antifungal agents include voriconazole, posaconazole and itraconazole. The resistance rate to fluconazole in this study in C. albicans was 4.9% with MIC90 value 2 μg/ml and ECV 8 μg/ ml, followed by C. tropicalis 8.8%, MIC90 value 4 μg/ml and ECV 0.5 μg/ml. The resistance rates in C. albicans to fluconazole were reported 12% (14/117) in colonizing isolates in neutropenic patients, 9.3% (16/354); and 81% (43/53) in infecting isolates (12)(13)(14). In Wisplinghoff et al. report 100% of C. glabrata, 4.9% of C. tropicalis, 2.9% of C. parapsilosis and 0.8% of C. albicans were not susceptible to fluconazole (18). The resistance rate of C. glabrata to fluconazole was reported 36% with 64% susceptible dose dependent (20). The acquired resistance to fluconazole (29.4%; P<0.05) is reported in C. glabrata isolates from colonized oral cavity in patients exposed to azoles (21). The increase resistance rate of Candida species to fluconazole maybe due to the frequent use of its medication. Voriconazole is an active azole antifungal agent against Candida species. In the present study, its susceptibility rate in C. glabrata and C. krusei, as the resistant Candida species, were 100%, (MIC50: 0.032 μg and MIC90: 0.016μg), and 92.3% (MIC50: 0.25 and MIC90: 0.5), respectively. The non-susceptible rates of Candida species to voriconazole were reported 9.8% of C. tropicalis, 7.6% of C. parapsilosis, 5.0% of C. krusei and 0.6% of C. albicans (18). In study done by pfaler et al., only C. krusei was resistant to voriconazole and other Candida species were susceptible to it (22). There is no breakpoint for posaconazole and ketoconazole, according to CISI M27 S4 (10). Posaconazole is the newest triazole antifungal and very expensive in our region. All Candida species had MIC value between 0.032 and 0.5 µg/ml. Candida glabrata MIC90 values for posaconazole and ketoconazole were 0.5 µg/ml and 0.125 µg/ml with GM 0.082 µg/ml and 0.037 µg/ ml, respectively. MIC90 value and GM for C. tropicalis to posaconazole and ketoconazole were 0.25 µg/ml and 0.25 µg/ml; and 0.035 µg/ml and 0.029 µg/ml, respectively. The MIC values for posaconazole were reported 0.016 µg/ml, 0.25 µg/ml, 0.125 µg/ml and 0.5 µg/ml in C. albicans, C. tropicalis, C. parapsilosis and C. glabrata, respectively (23). Posaconazole and voriconazole are used limitedly and are effective on Candida species isolates from the patients.
Of the echinocandin antifungal agents, caspofungin is more prescribed in our region. The resistant and intermediate rates of Candida species to caspofungin in this study were 0.4% and 2.7%; 2.9% and 5.9%; 18.8% and 47.9%; and 0.0% and 16.7% in C. albicans, C. tropicalis, C. glabrata and C. parapsilosis, respectively. Other Candida species were susceptible to caspofungin. C. krusei has intrinsic resistance to fluconazole and has been shown the highest sensitivity to caspofungin and voriconazole (S=93.3%). In Korean patient, none of the Candida species was resistant to caspofungin (25).

CONCLUSiON
Colonizing Candida species may be present as reservoir for future systemic candidiasis. In the present study, 49.2% of pediatric patients wit hematologic disorders were colonized with Candida species. C. albicans was the most prevalent species in pediatric colonized patients. New azole agents like voriconazole and posaconazole are effective to non-albicans Candida species. Increase in intermediate species is alarming to future emerging resistant species. The information about distribution and susceptibility patterns of species can be useful to appropriate treatment in hematopoietic pediatric patients at the duration of infection when sampling is impossible.